Tick-borne Apicomplexa in wildlife and ticks of French Guiana

Tick-borne Apicomplexa encompass a group of parasites responsible for significant medical and veterinary diseases, including babesiosis, theileriosis, and hepatozoonosis. In this study, we investigated the presence and diversity of tick-borne Apicomplexa in wildlife and ticks inhabiting the Amazon rainforests of French Guiana. To this end, we conducted molecular screening and typing using 18S rRNA sequences on a collection of 1161 specimens belonging to 71 species, including 44 species of wild mammals, five species of passerines, and 22 species of ticks. We characterized eight genovariants of Babesia, Theileria, Hemolivia, and Hepatozoon parasites, some matching known species, while others suggested potential novel species. These parasites were detected in wild mammals, including opossums, sloths, armadillos, porcupines, margays, greater grisons, and ticks, but not in passerines. Finally, similarities with surveys conducted in Brazil highlight the specific sylvatic transmission cycles of South American tick-borne Apicomplexa.

Tick-borne Apicomplexa are classified and taxonomically identified based on their morphological, biological, and molecular characteristics [22,23,38,43].Molecular phylogenies using the 18S rRNA gene sequences have recently challenged the previous taxonomic classification of piroplasmids, revealing that Babesia and Theileria each represent polyphyletic groupings within their respective orders [23].Hence, different Babesia species cluster with some Theileria species rather than forming cohesive monophyletic genera.However, phylogenetic approaches also indicate that each lineage of tick-borne Apicomplexa is typically associated with a restricted range of vertebrate host taxa, suggesting that these lineages have often evolved specializations to their respective vertebrate hosts [22,23,38,43].For instance, Babesia species of the Percei lineage exclusively infect birds, while species of the Cytauxzoon lineage are predominantly found in felids [23].Members of the lineage Babesia sensu stricto form a notable exception, as they infect multiple and diverse mammalian and avian vertebrate hosts [23].Nevertheless, surveys of wildlife continue to uncover new tick-borne Apicomplexa that do not fit into recognized species [4,5,13,20,21,26,30,31,34,36,43].Their host range, and phenotypic and developmental characteristics remain unknown in many cases [23,47].
In this study, we investigate the presence and diversity of tick-borne Apicomplexa in wild mammals, birds and ticks inhabiting the Amazon rainforests of French Guiana.This territory of 84,000 km 2 is predominantly covered by old-growth rainforests hosting one of the highest biodiversities in the world [42].While recent surveys have uncovered novel tick-borne microbes in French Guiana [7,11,12,25,32], including novel tick-borne pathogens infecting humans [14,19], there have been few studies focusing on tick-borne Apicomplexa in this region.In 1988, Babesia choloepi was described as a novel species in Linnaeus's two-toed sloths (Choloepus didactylus) of French Guiana based on morphological features [18], but no molecular sequences are available to complete its description.Furthermore, a survey of blood smears revealed the presence of several undetermined piroplasmid and Hepatozoon species in most mammalian orders of French Guiana, except primates, and in several species of snakes [41].However, none of these previous studies have characterized these tick-borne Apicomplexa based on molecular typing.

Ethics
Mammal samples were collected and used in accordance with an international CITES permit (Convention on International Trade in Endangered Species of Wild Fauna and Flora; permit FR973A) following French legislation.Following sharing policies in French Guiana, mammal samples are registered in the JAGUARS collection (https://kwata.net/gestioncollection-biologique;CITES reference: FR973A) supported by Kwata NGO (accredited by the French Ministry of the Environment and the Prefecture of French Guiana, Agreement R03-2019-06-19-13), Institut Pasteur de la Guyane, Direction Générale des Territoires et de la Mer (DGTM), Collectivité Territoriale de la Guyane, and validated by the French Guianese prefectural decree No. 2012/110.The French Ministry of Higher Education and Research provides authorization for projects using wild animals for scientific purposes (reference APAFIS-37571-2022111610578451).Permits for bird sampling (French Guiana prefectural decrees Nos.2011/003, 2013/127 and R03-2018-10-30-0092) authorized the capture, marking, sampling, holding and transport of bird samples.Bird sampling was also done with permissions from several organizations: the DGTM de Guyane, the Direction Régionale de l'Office National des Forêts (ONF) de Guyane, the Conservatoire du Littoral, the Centre National d'Études Spatiales (CNES), the Centre Spatial Guyanais (CSG), the Association pour la Découverte de la Nature en Guyane, the association Randoroura.The use of bird genetic resources is declared to the French Ministry of the Environment under reference TREL1820249A/49 in accordance with the Nagoya Protocol on Access and Benefit Sharing (ABS).The French Ministry of the Environment also validated the collection and use of tick samples under the reference TREL19028117S/156, in accordance with the ABS Nagoya Protocol.All animals were handled in strict accordance with good animal practice and ethical standards as defined by the French code of practice for the care and use of animals for scientific purposes, established by articles R214-87 to R214-137 of the French rural code.

Collection of samples
We used archived DNA templates extracted from 1161 specimens collected in French Guiana between 1994 and 2019 (Tables 1 and S1).This collection comprises samples from 71 species, including 44 species of wild mammals (n = 626 samples), five species of passerines (n = 247), and 22 species of ticks (n = 288).It includes blood samples (for wild mammals, and passerines), spleen samples (wild mammals), or whole body (ticks).We had primarily collected these samples as part of previous studies among wildlife, and ticks in French Guiana [7-10, 12, 14].

Molecular detection and typing of tick-borne Apicomplexa
Each DNA template underwent individual testing using semi-nested polymerase chain reaction (PCR) targeting a fragment of the 18S rRNA (SSU) gene to detect Babesia, Theileria, Cytauxzoon, Hemolivia, and Hepatozoon (Table S2).Seminested PCR amplifications were performed as follows: the first PCR run with the external primers was performed in a 10 lL volume containing 10-50 ng of genomic DNA, 3 mM of each dNTP (Thermo Scientific, Waltham, MA, USA), 8 mM of MgCl2 (Roche Diagnostics), 3 lM of each primer, 1 lL of 10Â PCR buffer (Roche Diagnostics), and 0.5 U of Taq DNA polymerase (Roche Diagnostics).A 1 lL aliquot of the PCR product from the first reaction was then used as a template for the second round of amplification.The second PCR was performed in a total volume of 25 lL and contained 8 mM of each dNTP (Thermo Scientific), 10 mM of MgCl2 (Thermo Scientific), 7.5 lM of each of the internal primers, 2.5 lL of 10ÂPCR buffer (Thermo Scientific), and 1.25 U of Taq DNA polymerase (Thermo Scientific).Positive (DNA templates of Dermacentor marginatus ticks collected in the South of France and confirmed positive for Babesia bovis) and negative (water) controls were included in each PCR assay.Following visualization via electrophoresis in 1.5% agarose gel, positive PCR products were sequenced by Eurofins.Sequence chromatograms were cleaned with Chromas Lite (http://www.technelysium.com.au/chromas_lite.html).New sequences obtained in this study are available in GenBank under accession numbers PP476856-PP476867.

Molecular phylogenetic analyses
Phylogenetic analyses were based on alignments of the Apicomplexa 18S rRNA gene sequences using MAFFT (https://mafft.cbrc.jp)for Piroplasmida and Eucoccidiorida, respectively.Sequences of Apicomplexa obtained from Gen-Bank, including representative species of the genera Babesia, Theileria, Cytauxzoon, Hemolivia, and Hepatozoon were also included in the phylogenetic analyses.The Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/blast/Blast.cgi) was used to find 18S rRNA gene sequences available on GenBank and showing the highest nucleotide similarities with the gene sequences we characterized in this study.The Gblocks program with default parameters was used to obtain non-ambiguous sequence alignments [16].Phylogenetic analyses were performed using maximum-likelihood (ML) analyses on both alignments using the MEGA software package (https://www.megasoftware.net/).The evolutionary models that best fit the sequence data were determined using the Akaike information criterion.Clade robustness was assessed by bootstrap analysis using 1000 replicates.

Detection of tick-borne Apicomplexa
Concerning the 71 species examined, molecular survey led to the identification of tick-borne Apicomplexa in 11 species (Tables 1 and S1).They were detected in seven species of mammals, including the common opossum (Didelphis marsupialis), gray four-eyed opossum (Philander opossum), Linnaeus's two-toed sloth (Choloepus didactylus), nine-banded armadillo (Dasypus novemcinctus), porcupine (Coendou sp.), margay (Felis wiedii), and greater grison (Galictis vittata).They were also detected in four tick species, including the two species that most commonly bite humans in South America, the Cayenne tick (Amblyomma cajennense) and Amblyomma oblongoguttatum.The other two infected species were the opossum tick (Ixodes luciae), and the iguana tick (Amblyomma dissimile).No parasites were detected in the five species of passerines surveyed.
Similarly, the Hepatozoon genovariant #3 was present in margay and A. oblongoguttatum, a tick species feeding on a wide range of mammals.The other six genovariants were each found associated with only one species, either mammals or ticks (Table S3).Three of the eight genovariants showed 100% nucleotide identity for their 18S rDNA sequences with known protozoan species (Table S3).Hemolivia genovariant #1, identified in A. issimile, is identical to Hemolivia stellata, a parasite of the cane toad Rhinella marina [37].Hepatozoon genovariant #3, detected in margay and A. oblongoguttatum, matched to Hepatozoon luiperdjie, a recently described parasite of the African leopard [6].Hepatozoon genovariant #5, found in A. dissimile, matched to Hepatozoon cuorae, a parasite of Asian reptiles [43].While Hemolivia stellata is native to South America [37], neither Hepatozoon luiperdjie nor Hepatozoon cuorae have been previously observed in the Americas [43].The five other genovariants are distinct from taxa referenced in public databases based on their 18S rDNA sequences (Table S3).

Phylogeny of tick-borne Apicomplexa
ML phylogenetic analyses based on 18S rRNA nucleotide sequences were further conducted to examine the phylogenetic proximity of tick-borne Apicomplexa detected in this study with representative species of the genera Babesia, Theileria, Cytauxzoon, Hemolivia, and Hepatozoon.ML analyses confirmed that (i) the two genovariants of Babesia (#7 and 8) and the genovariant of Theileria (#6) cluster with other piroplasmids (Fig. 1), (ii) the four genovariants of Hepatozoon (#2, 3, 4, and 5) and the genovariant of Hemolivia (#1) cluster with other Hepatozoon and Hemolivia species (Fig. 2).As expected [23], Piroplasmida is formed by the genus Cytauxzoon and a polyphyletic assemblage of Babesia and Theileria species.
ML analyses further showed that the Babesia genovariants #7 and #8 and the Theileria genovariant #6 are distantly related within the Order Piroplasmida (Fig. 1).Babesia genovariant #7 from Linnaeus's two-toed sloths does not belong to a previously identified piroplasmid clade, including the recently described clades found in wildlife in Brazil, as the Phyllostomus bat clade [21], the Tapirus clade [30], the South American Marsupialia Group clade [31] and the Capybara clade [26].Babesia genovariant #8 from A. cajennense belongs to the lineage Babesia sensu stricto (clade X in [23]), and is related to Babesia vogeli, one of the most important pathogens among Babesia species found in dogs [33].Theileria genovariant #6 from nine-banded armadillos belongs to the Theileria sensu stricto lineage (clade IX in [23]), which include causative agents of tropical theileriosis, such as Theileria orientalis and Theileria annulata, two parasite species usually found in cattle [1].In the Theileria sensu stricto lineage, Theileria genovariant #6 is closely related to another undescribed Theileria species recently found in nine-banded armadillos in Brazil (Fig. 1) [15,39].
For hemogregarines, ML analyses confirmed the relatedness of Hemolivia genovariant #1 with H. stellata, and further indicated that they are both related to Hemolivia parvula, Hemolivia mauritanica, and Hemolivia mariae (Fig. 2), infecting African, Palearctic, and Australian reptiles, respectively [28,50].Hepatozoon genovariant #2 found in opossums, porcupines and the opossum tick I. luciae forms a branch that is well divergent from all other Hepatozoon species, with no close relatedness to known species or isolates.Hepatozoon genovariant #3 clusters with Hepatozoon luiperdjie, and both are closely related to the common cat parasite Hepatozoon felis.The Hepatozoon genovariant #4 we detected in greater grison is more closely related to Hepatozoon canis and Hepatozoon americanum, which infect dogs in the Americas.Hepatozoon genovariant #5 clusters with Hepatozoon cuorae and Hepatozoon colubri, which both infect turtles and snakes (Fig. 2) [44,48,49].

Discussion
In our study, we have identified several genovariants of Babesia, Theileria, Hemolivia, and Hepatozoon in various wildlife species, including opossums, sloths, armadillos, and porcupines, as well as in tick species that most commonly bite humans in South America.While some genovariants match with known species, others do not, raising evidence of undescribed Apicomplexa species in French Guiana.The detection of similar genovariants in wildlife and ticks further unveils the existence of unique sylvatic transmission cycles for these Apicomplexa.These observations in French Guiana remarkably mirror recent surveys of wildlife in Brazil, which also highlight the distinctiveness of South American tick-borne Apicomplexa and their specific sylvatic transmission cycles [4,5,13,20,21,26,30,31,34,36,43].
None of the three piroplasmid genovariants found in French Guiana matched with Babesia, Theileria, and Cytauxzoon species, strains or isolates for which 18S rRNA sequences were available in public databases.They may each represent a new putative species or an already described species for which no molecular sequences are currently available.Indeed, Babesia choloepi, which was morphologically characterized from erythrocytes of Linnaeus's two-toed sloths of French Guiana [18], may be the Babesia genovariant #7 we characterized also in Linnaeus's two-toed sloths.This Babesia genovariant is distinct from all already known piroplasmid clades, including those recently described in wildlife in Brazil [21,26,30,31], suggesting it may form a novel piroplasmid clade.The Theileria genovariant #6 of the nine-banded armadillo is similar to a Theileria sp.infected in nine-banded armadillos in Brazil [15,39], and both may be related to another Theileria sp., observed through microscopy in nine-banded armadillos in the state of Pará, Brazil, as mentioned by Lainson et al. [29], but not further described.The association of armadillos with the Theileria genovariant #6 is more singular since this parasite is related to species of the Theileria sensu stricto lineage and that are usually found in cattle [23].The Babesia genovariant #8 is closely related to Babesia vogeli, which is commonly found in dogs and cats in Brazil [4].Its detection in A. cajennense, a tick species feeding on a wide range of mammals, including humans, suggests that a diverse range of hosts is exposed to this parasite.However, Babesia genovariant #8 has never been detected in vertebrates, indicating that its natural hosts are currently unknown.
The diversity of Hemolivia and Hepatozoon genovariants found in French Guiana leads to quite similar observations.Hemolivia genovariant #1 is evidently Hemolivia stellata, a parasite that colonizes erythrocytes, cells of the reticuloendothelial system, and digestive cells of the cane toad [37].However, while A. rotundatum is vector of this parasite in Brazil [37], we commonly observed infection in another tick species in French Guiana, the iguana tick A. dissimile.This tick species specializes in reptiles and amphibians, commonly feeding on cane toads, and may thus serve as an additional vector species of Hemolivia stellata in French Guiana.Similarly, Hepatozoon genovariant #5 is related to Hepatozoon cuorae, a species known to infect Asiatic turtles [49].However, Hepatozoon cuorae has never been observed in South America, but our observation suggests that a related species may exist in French Guiana.Amblyomma dissimile is a potential local vector, and given the host specificity of this tick species, this parasite may infect South American reptiles or amphibians or both.Hepatozoon genovariant #3 is related to Hepatozoon luiperdjie, a parasite of the African leopard [6], previously not found outside Africa.The detection of this genovariant in margays of French Guiana, and in A. oblongoguttatum as a potential local vector, suggests that the Hepatozoon luiperdjie relatives may form a clade specific to wild felids across different continents.Hepatozoon genovariant #4 found in greater grison is different to all known species but is related to Hepatozoon canis and Hepatozoon americanum, forming together a large clade associated with carnivores.On the contrary, the discovery of Hepatozoon genovariant #2 in two opossum species, porcupines, and the opossum tick I. luciae indicates that this parasite is common and widespread in the rainforests of French Guiana.However, Hepatozoon genovariant #2 has not been documented prompting inquiries into its geographic distribution.conclusion, investigation into tick-borne Apicomplexa in French Guiana underscores the significance of acknowledging the diversity of these infections within this region.Moreover, the of potential novel species emphasizes urgent need for ongoing research and surveillance to mitigate the health risks posed by these tick-borne parasites.A major limitation of our study is that we used a short fragment of the 18S rDNA for the typing of infection.Complete sequencing of the 18S rDNA and additional markers, such as mitochondrial markers, are now needed to confirm the existence of new species and new clades.Notably, these new typings should allow for better comparison with the new species discovered in South America, particularly for Hepatozoon, for which many new genovariants have been described [2,3,13,35,40].

Figure 1 .
Figure 1.Phylogeny of piroplasmids (Piroplasmida order) constructed using maximum-likelihood (ML) estimations and based on 18S rDNA sequences (408 unambiguously aligned bp; best-fit approximation for the evolutionary model on the basis of Akaike information criterion: TN93+G+I).Only one 18S rDNA sequence per genovariant and per host species is shown for data produced in this study (in bold).GenBank accession numbers of sequences used in analyses are shown on the phylogenetic trees.Numbers at nodes indicate bootstrap support percentage with 1000 replicates.Only bootstrap values >70% are shown.The scale bar is in units of mean number of substitutions/site.The right part of the figure presents the prevalence of Apicomplexa infection observed for each infected species (on a logarithmic scale).

Figure 2 .
Figure 2. Phylogeny of tick-borne hemogregarines (Eucoccidiorida order) constructed using maximum-likelihood (ML) estimations based on 18S rDNA sequences (461 unambiguously aligned bp; best-fit approximation the evolutionary model on the basis Akaike criterion: HKY+G).Only one 18S rDNA sequence per genovariant and per host species is shown for data produced in this study (in bold).accession numbers of sequences used in analyses are shown the phylogenetic trees.Numbers at nodes indicate bootstrap support percentage with 1000 replicates.Only bootstrap values >70% are shown.The scale bar is in units of mean number of substitutions/site.The right part of the figure presents the prevalence of Apicomplexa infection observed for each infected species (on a logarithmic scale).

Table 1 .
et al.: Parasite 2024, 31, 49 List of mammal, passerine and tick species examined for the presence of tick-borne Apicomplexa in French Guiana.A detailed list of specimens and sampling locations is provided in TableS1.